S. Anne Böttger

p53 Superfamily Proteins in Marine Bivalve Cancer and Stress Biology

The human p53 tumour suppressor protein is inactivated in many cancers and is also a major player in apoptotic responses to cellular stress. The p53 protein and the two other members of this protein family (p63, p73) are encoded by distinct genes and their functions have been extensively documented for humans and some other vertebrates. The structure and relative expression levels for members of the p53 superfamily have also been reported for most major invertebrate taxa. The functions of homologous proteins have been investigated for only a few invertebrates (specifically, p53 in flies, nematodes and recently a sea anemone). These studies of classical model organisms all suggest that the gene family originally evolved to mediate apoptosis of damaged germ cells or to protect germ cells from genotoxic stress. Here, we have correlated data from a number of molluscan and other invertebrate sequencing projects to provide a framework for understanding p53 signalling pathways in marine bivalve cancer and stress biology. These data suggest that (a) the two identified p53 and p63/73-like proteins in soft shell clam (Mya arenaria), blue mussel (Mytilus edulis) and Northern European squid (Loligo forbesi) have identical core sequences and may be splice variants of a single gene, while some molluscs and most other invertebrates have two or more distinct genes expressing different p53 family members; (b) transcriptional activation domains (TADs) in bivalve p53 and p63/73-like protein sequences are 67-69% conserved with human p53, while those in ecdysozoan, cnidarian, placozoan and choanozoan eukaryotes are ≤33% conserved; (c) the Mdm2 binding site in the transcriptional activation domain is 100% conserved in all sequenced bivalve p53 proteins (e.g. Mya, Mytilus, Crassostrea and Spisula) but is not present in other non-deuterostome invertebrates; (d) an Mdm2 homologue has been cloned for Mytilus trossulus; (e) homologues for both human p53 upstream regulatory and transcriptional target genes exist in molluscan genomes (missing are ARF, CIP1 and BH3 only proteins) and (f) p53 is demonstrably involved in bivalve haemocyte and germinoma cancers. We usually do not know enough about the molecular biology of marine invertebrates to address molecular mechanisms that characterize particular diseases. Understanding the molecular basis of naturally occurring diseases in marine bivalves is a virtually unexplored aspect of toxicoproteomics and genomics and related drug discovery. Additionally, increases in coastal development and concomitant increases in aquatic pollutants have driven interest in developing models appropriate for evaluating potential hazardous compounds or conditions found in the aquatic environment. Data reviewed in this study are coupled with recent developments in our understanding the molecular biology of the marine bivalve p53 superfamily. Taken together, they suggest that both structurally and functionally, bivalve p53 family proteins are the most highly conserved members of this gene superfamily so far identified outside of higher vertebrates and invertebrate chordates. Marine bivalves provide some of the most relevant and best understood models currently available for experimental studies by biomedical and marine environmental researchers.

Cytoplasmic sequestration of the tumor suppressor p53 by a heat shock protein 70 family member, mortalin, in human colorectal adenocarcinoma cell lines

While it is known that cytoplasmic retention of p53 occurs in many solid tumors, the mechanisms responsible for this retention have not been positively identified. Since heatshock proteins like mortalin have been associated with p53 inactivation in other tumors, the current study sought to characterize this potential interaction in never before examined colorectal adenocarcinoma cell lines. Six cell lines, one with 3 different fractions, were examined to determine expression of p53 and mortalin and characterize their cellular localization. Most of these cell lines displayed punctate p53 and mortalin localization in the cell cytoplasm with the exception of HCT-8 and HCT116 379.2 cells, where p53 was not detected. Nuclear p53 was only observed in HCT-116 40-16, LS123, and HT-29 cell lines. Mortalin was only localized in the cytoplasm in all cell lines. Co-immunoprecipitation and immunohistochemistry revealed that p53 and mortalin were bound and co-localized in the cytoplasmic fraction of four cell lines, HCT-116 (40-16 and 386; parental and heterozygous fractions respectively of the same cell line), HT-29, LS123 and LoVo, implying that p53 nuclear function is limited in those cell lines by being restricted to the cytoplasm. Mortalin gene expression levels were higher than gene expression levels of p53 in all cell lines. Cell lines with cytoplasmic sequestration of p53, however, also displayed elevated p53 gene expression levels compared to cell lines without p53 sequestration. Our data reveal the characteristic cytoplasmic sequestration of p53 by the heat shock protein mortalin in human colorectal adenocarcinoma cell lines, as is the case for other cancers, such as glioblastomas and hepatocellular carcinomas.

Selective initiation and transmission of disseminated neoplasia in the soft shell clam Mya arenaria dependent on natural disease prevalence and animal size

Disseminated neoplasia, a diffuse tumor of the hemolymph system, is one of the six most destructive diseases among bivalve mollusk populations, characterized by the development of abnormal, rounded blood cells that actively proliferate. Though the specific etiology of disseminated neoplasia in Mya arenaria remains undetermined, the involvement of viral pathogens and/or environmental pollutants has been suggested and considered. The current study used 5-bromodeoxyuridine (BrDU) known to induce the murine leukemia virus and filtered neoplastic hemolymph to initiate disseminated neoplasia in clams from different populations and size classes respectively. M. arenaria from three locations of different natural neoplasia occurrences were divided into a control and three experimental treatments and injected with 200 μl of sterile filtered seawater or 50-200 μg/ml BrDU respectively. In a concurrent experiment, animals from different size classes were injected with 2.5% total blood volume of 0.2 μm filtered blood from a fully neoplastic animal. Animals were biopsied weekly and cell neoplasia development was counted and scored as 0-25, 26-50, 51-75 and 76-100% neoplastic hemocytes (stages 1-4) in 50 μl samples. BrDU injection demonstrated that neoplasia development in M. arenaria was dose dependent on BrDU concentration. In addition, natural disease prevalence at the source location determined initiation of neoplasia induction, with animals from the area of the highest natural disease occurrence displaying fastest neoplasia development (p=0.0037). This could imply that depending on the natural disease occurrence, a potential infectious agent may remain dormant in normal (stage 1) individuals in higher concentrations until activated, i.e. through chemical injection or potentially stress. The size experiment demonstrated that only M. arenaria between 40 and 80 mm developed 26-100% neoplastic hemocytes when injected with filtered neoplastic hemolymph, indicating that individuals smaller than 20mm or larger than 80 mm were not or no longer susceptible to disease development. So far neoplasia studies have not considered natural disease prevalence or size involvement in neoplasia development and our results indicate that these should be future considerations in neoplasia examinations.

Care and Maintenance of Adult Echinoderms

Publisher Summary This chapter addresses the care and maintenance of adult echinoderms that produce gametes and embryos commonly used for molecular, cellular, and developmental biology. Emphasis is on sea urchin and sea star species from North and South America, Europe, and Japan. The chapter discusses adult echinoderm models: (1) reproductive cycles and (2) gametogenesis. Annually, within the gonads of both sexes of most echinoderms, millions of gonial cells (oogonia and spermatogonia) originate by mitosis, produce gonocytes (primary and secondary) that undergo gametogenesis and result in gametes that are stored. The methods for obtaining adult echinoderms are also discussed. Echinoderms are highly sensitive to changes in their environment and are handled very carefully during collection. Echinoderms collected by scuba survive longer than those collected by mechanical dredging. The maintenance of sea urchins in small-scale, closed systems where water is recirculated is described for different species of sea urchins. Two designs for land-based sea urchin aquaculture systems discussed in the chapter are the toboggan and trough systems, along with methods for the care and handling of adult echinoderms.

Chronic Natural Occurrence of Disseminated Neoplasia in Select Populations of the Soft-Shell Clam, Mya arenaria, in New England

Abstract Disseminated neoplasia, a diffuse tumor of the hemic system, is characterized in many bivalve mollusks by hemolymph containing 1–100% mitotic hemocytes. Little is known about the onset and chronic distribution of neoplasia in populations of Mya arenaria (Soft-shell Clam), though studies have reported episodic exposure to environmental contaminants or an infectious agent as a potential cause of this disease. Here we provide the first set of continuous data on neoplasia in Soft-shell Clams, from three sites in New England where sediments have been characterized regarding their granulometry, composition, contaminants, and clam densities. When correlating sediment characteristics to terminal neoplasia (76–100% neoplastic or rounded, unattached hemocytes), New Bedford Harbor, MA, which is the most contaminated site, had the highest frequency of treminal neoplasia (maximum of 9.49% ± 0.78 SE), and the most pristine site, Ogunquit, ME, displayed the lowest frequencies (maximum of 0.47% ± 0.05 SE). Correlations of frequency of neoplasia to known environmental contaminants also suggests that fully neoplastic individuals were found only at sites of increased levels of heavy metals, PCBs, and PAHs. In addition, we documented the highest frequency of clams with terminal neoplasia from New Bedford Harbor in December (9.49% ± 0.78 SE) when seawater temperatures were low, and the lowest frequency in July (1.08 ± 0.4 SE) when seawater temperatures were highest. These results may indicate vulnerability of neoplastic clams to seasonal increases in environmental temperature and resulting oxidative stress. Based on shell measurements and a theoretical mathematical age model (which correlates susceptibility to neoplasia with age and sexual maturity), we suggest that the Soft-shell Clam is most susceptible to this disease between one and two years of age (9.5% frequency at 1 year, 22.25% incidence at 1.5 years, and 57.14% incidence at 2 years).

Mortalin in Invertebrates and The Induction of Apoptosis by Wild-Type p53 Following Defeat of Mortalin-Based Cytoplasmic Sequestration in Cancerous Clam Hemocytes

In this chapter we demonstrate the remarkable sequence homology between mortalin proteins from a broad array of invertebrate taxa, including the few species for which functional analyses have been conducted. We also discuss expression and functional data for full-length and truncated clam homologs for human mortalin and their function in cytoplasmic sequestration in cancerous clam hemocytes. Both clam proteins have N-terminal mitochondrial targeting and p53 binding domains, though the truncated variant is missing exon 3 containing the N-terminal ATP/ADP binding and ATPase domains. Both variants are over-expressed and complexed with p53 and both may be responsible for cytoplasmic sequestration of p53 in cancerous clam hemocytes. Clam hemocyte cancer is the only animal model thus far investigated where cytoplasmically sequestered wild-type p53 can be reactivated both in vitro and in vivo using both genotoxic and non-genotoxic therapies. Our results suggest that mortalin-based cytoplasmic sequestration of wild-type p53 in cancerous clam hemocytes can be reversed by treatment with antineoplastic drugs also employed against similar human diseases and will result either in transcription-based apoptosis when the nucleus is accessible or non-transcription-based apoptosis when nuclear access is blocked.

Morphology and microanatomy of harbor porpoise (Phocoena phocoena) dorsal fin tubercles.

The unique pattern of small tubercles on the leading edge of the dorsal fins of harbor porpoises (Phocoena phocoena) has been widely noted in the literature, though their structure or function has never been conclusively identified. We examined external morphology and microanatomy of the tubercles for further understanding of the nature of the tubercles. Measurements were taken of height and peak‐to‐peak distance of the tubercles using scaled photographs. Mean tubercle height was standardized as a percentage of the dorsal fin height and ranged from 0.63 to 0.87%. Mean peak‐to‐peak distance ranged from 4.2 ± 2.0 to 5.6 ± 2.0 mm. The microstructure analysis of the dorsal fin leading edge, trailing edge and tubercles revealed an epidermal thickness of 0.7–2.7 mm with the thickest epidermis at the tubercular apex. The epidermis contained three distinct strata (=layers), including the stratum corneum, spinosum, and basale. The stratum corneum was significantly thickened in tubercles, over four times thicker than in the leading or trailing edge of the fin. The stratum spinosum, composed of lipokeratinocytes and lamellar oil bodies, was significantly thinner in the trailing edge than in the other two sites. There was no significant difference in the stratum basale among the three sites. Volume fraction of lipokeratinocytes was significantly higher at the sides of the leading edge and the apex of the tubercles, while volume fraction of lamellar oil bodies was significantly lower at the apex of the tubercles. Though the function of the tubercles is unknown, their position, hardened structure and increased epidermal stratum corneum suggest that they may have hydrodynamic importance. J. Morphol., 2011.

Ecology Content in Introductory Biology Courses: a Comparative Analysis

ABSTRACT In recent years the need for ecological literacy and problem solving has increased, but there is no evidence that this need is reflected by increased ecology coverage at institutions of higher education (IHE) across the United States. Because introductory biology courses may serve to direct student interest toward particular biological categories such as ecology, time devoted to topics in these categories within introductory biology courses may be crucial for captivating student interest. In a 2009 survey, members of the National Association of Biology Teachers (NABT) College and University Sections identified 20 topics they considered essential for inclusion in introductory biology courses. The NABT members, acknowledging the importance of ecological concepts, considered two ecological topics essential. The present study evaluated the actual coverage of ecology and other topic categories compared to recommendations and according to location. For this purpose, lecture and lab syllabi were collected from 26 rural, suburban, and urban IHEs from the Mid-Atlantic region. Course content was divided into eight categories, including ecology, and percentages of total lecture and lab time per category were calculated. This actual coverage was compared to the NABT recommendations. Actual coverage of ecology was not significantly different from coverage recommended by the NABT members, whereas cell/molecular/biochemistry and evolution were lower and genetics, development, and taxonomy were higher than recommended. Course content was also compared by location, with no significant effect of institutional location on ecology coverage. We conclude that although students taking introductory biology courses in Mid-Atlantic IHEs are likely to receive the NABTs recommended coverage of ecology instruction regardless of institutional location, actual ecology coverage has not increased, regardless of the increased need for ecological literacy.

Survey for the presence of a vitronectin-like protein in micro- and macroalgae and cyanobacteria

Vitronectin (Vn) is a glycoprotein that serves a wide variety of roles in multicellular organisms. It was first identified in multicellular animals but has also been isolated from land plants and some algae, where it appears to serve as an extracellular adhesive molecule. In order to further elucidate presence and localization of a Vn–like protein and its potential role in algae, we surveyed different morphological regions of 24 species of macro‐ and microalgae and three species of cyanobacteria for the presence of a Vn–like protein. Vn–like proteins were not detected in any of the species of cyanobacteria, microalgae or Rhodophyta investigated. They were detected in several species of the Phaeophyceae and Chlorophyta where their localization was limited to the holdfast and rhizoids of these organisms, respectively. Detection of a Vn–like protein (between 0.0125 and 0.097 μg · μL−1 protein extract) was therefore limited to locations associated with substrate attachment.